With the increased prevalence of multidrug resistant bacterial pathogens such as enterococci resistant to vancomycin (VRE), we have been engaged in studies to enhance the therapeutic efficacy of bacterial viruses or bacteriophages (phages). Most phages are very specific to the species or strain of bacteria. Although this specificity may limit the usefulness of phage therapy, it may be clinically useful, because such phages can be targeted to the specific infectious bacteria and will not affect the normal flora of the body. However in certain situations such as in urinary tract infections with strains of E. coli. it would be useful to have a single phage that could affect more than one pathogenic strain to address this problem. We characterized E. coli phages that are specific for certain pathogenic K antigen strains. In this group of phages, host range is determined by presence of specific hydrolytic tail proteins. We discovered that if a phage contains genes for more than one specific hydrolytic tail protein then that phage will be infective for each strain of E. coli. Such phage are polyvalent in their capacity to infect more than one strain of bacteria, expressing an outer capsule that can serve as a substrate for that hydrolytic enzyme found on the phage tail. This discovery allows us to develop additional phage with extended host ranges. A patent has been filed concerning this endeavor and manuscripts have been published in J. Virology and J. Bacteriology. We have now completed the genome sequence of one of these phages, K1-5, and are working on a manuscript involving comparative genomics of this phage and a similar phage, SP6, in collaboration with Dr. Ian Molineux of the University of Texas. In addition, we have isolated and characterized several other phages with different host specificities which we are currently sequencing to determine the mode of host specificity. An NIAID biodefense proposal for intramural biodefense research has been submitted to study bacteriophage therapy for antibiotic resistant Y. pestis (plague) and to develop phage based detection methods for plague. An invited chapter on the use of phage for antibacterial therapy for the book "The Bacteriophage" has been completed. In addition, given the importance of rapidly determining whether a particular phage will be effective as an antibacterial therapeutic agent, we are currently developing phage that express reporter genes. Prior to this effort, determination was performed by plaque assays that can take from 12 hours to several days to develop. We are currently developing methods for incorporating reporter genes, such as b-galactosidase or luciferase genes into phages that may be of clinical use as antibacterial agents. An invention report concerning these efforts has been submitted. Previous studies in this laboratory demonstrated that phage administered to animals was rapidly removed by the host defense systems, particularly the organs of the reticuloendothelial system (RES). To reduce phage elimination by the host defense system, we developed a serial passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of phage for a number of different species of bacteria. We also demonstrated that these long-circulating phage mutants have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Additional factors known to have impeded therapeutic antibacterial applications of phage included: the failure to recognize the relatively narrow host range of phages and the presence of toxins in unpurified phage. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. In these efforts we have isolated bacteriophage with activity against enterococci resistant to vancomycin (VRE). The emergence of VRE poses a problem for patients with immune deficiency related illnesses (including those who have been immune suppressed for organ transplantation). From a clinical point of view, there are currently few therapeutic agents commercially available with established efficacy for patients infected by VRE. We tested the ability of phage to rescue mice infected with lethal doses of a strain of VRE isolated from a human infection. Preliminary studies demonstrated a dose response for the phage titers used to rescue VRE infected mice. Experiments were also performed that demonstrate that phage could be used to rescue mice late in the course of a VRE illness. A manuscript concerning this work has been published in Infection and Immunity. Although the mouse experiments do not completely mimic the course of human illness with VRE, primarily because we administered a larger lethal dose of bacteria than most humans are confronted with early in their disease. Despite this difference we were encouraged by the fact that we were able to rescue significant numbers of mice late in the infectious cycle. Two additional concerns have confronted those interested in the use of phage as an antibacterial agent. The first, has been a belief that bacteria will quickly develop resistance to phage. However, in a comparative study of mice given potentially lethal intramuscular or intracereberal injections of bacteria, a single intramuscular dose of phage was more effective than multiple intramuscular injections of: tetracycline, ampicillin, choramphenicaol or trimethoprim plus sulphafuraxole. The authors of this study, Smith and Huggins, noted that the therapeutic success of phage was due to its high in vivo activity and the failure of phage-resistant mutants to proliferate during treatment. Levin and Bull repeated this study with similar results and they concluded that the exponential growth of the phage also reduced the appearance of resistant stains of bacteria. The second concern is that the FDA would never approve the use of a virus to treat bacterial infections. In this regard it should be noted that a phage, phiX174, has been approved as a marker for assessing the immune response in AIDs and other immune deficient patients. In this application phiX174 is injected intravenously and serum is monitored for the presence of phage and antibody production against the phage. The Laboratory is maintaining the Phage-Tech Interest Group (PhTIG), and is involved in the establishment of a phage applications web site: http://www.nih.gov/sigs/phtig/. Two of the lab members, C.M. and D.S. have been invited to the meeting Phage Therapy-Potential and Challenges at the Banbury Center, Cold Spring Harbor Laboratories in November 2002, and we have organized a session for the 103rd annual meeting of the American Society for Microbiology in D.C. in May 2003.